Mucosal atrophy often leads to barrier failure and sepsis in starving or fasting patients despite total parenteral nutrition (TPN). Many patients who undergo massive bowel resection fail to adapt and require permanent TPN with high mortality. Current interventions to prevent mucosal atrophy or enhance adaptation have limited efficacy, perhaps because they mostly induce proliferation. We hypothesize that hyperproliferation can be maladaptive as it is often accompanied by decreased differentiation. Thus, we propose that we must learn to promote differentiation as well as proliferation to achieve an intact functional mucosa, reverse mucosal atrophy, and engender maximal intestinal adaptation. Schlafen (Slfn)-family proteins mediate cell growth, differentiation, or development in hematopoietic cells in a Slfn and cell-specific fashion. We showed that the rodent protein Slfn3 is induced during and required for intestinal epithelial differentiation in response to various stimuli. We further found that Slfn3 varies with intestinal development and mucosal atrophy and promotes differentiation in vivo and that SLFN12 is its human ortholog. We hypothesize that Slfn3/12 induction represents a fundamental and essential common pathway for enterocytic differentiation that can be specifically targeted to promote differentiation and maintain the mucosa. We further propose that Slfns act in the cytosol through Slfn P-loop domain binding to specific proteins lowering proteasomal activity, increasing Cdx2 homeobox protein levels and enhancing transcription via a Cdx2-dependent positive feedback mechanism. We will study 1) intestinal mucosa in patients fasting on TPN (atrophy) or after Roux-en-Y gastric bypass (hypertrophy), 2) rat and human intestinal epithelial cells in vitro, and 3) the effects of in vivo manipulation of Slfn3/12 levelsin Slfn3 knock-out mice after 60% massive small bowel resection and a novel rodent model of intestinal epithelial atrophy and hypertrophy to: 1) Demonstrate that Slfn3 determines enterocytic differentiation in mucosal atrophy and adaptation in rodents, 2) Show that SLFN12 is the relevant human ortholog of Slfn3, and 3) Establish the mechanism(s) by which Slfn3/SLFN12 act. This study will fill a critical knowledge gap because we will delineate a fundamental mechanism by which diverse stimuli regulate intestinal epithelial differentiation. This will facilitate interventions to maintain the mucosal barrier in starving or critically ill paients with mucosal atrophy and promote nutrition in pediatric or adult short bowel syndrome. The Slfn3 knock-out mouse will provide valuable insights not only into the convergent differentiation regulatory pathway we will study but also into normal gut development, mucosal healing and other intestinal disorders such as IBD. In addition, understanding how Slfn proteins promote differentiation will have broader implications for differentiation and development in other epithelial tissues.